Granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) has replaced traditional bone marrow harvest as the preferred source of cells for hematopoietic stem cell transplantation. Though generally considered to be a safer alternative to harvest, there are increasing observations that G-CSF administration causes vascular and inflammatory complications due to enhanced adhesion of leukocytes to vascular endothelium. A major mediator of leukocyte-endothelial adhesive interactions is E-selectin, an inducible endothelial molecule that binds sialofucosylated carbohydrate antigens recognized by the mAb HECA-452. We reasoned that vascular and inflammatory complications may result from G-CSF-induced expression of E-selectin ligands on circulating leukocytes. We have found that P-selectin glycoprotein ligand-1 (PSGL-1) and Hematopoietic Cell E-and L-selectin Ligand (HCELL) are E-selectin ligands on G- CSF MPB leukocytes. Importantly, our studies also reveal that a novel HECA-452-reactive ~65kDa high affinity E-selectin ligand (hereinafter called "~65kDa protein") is expressed on G-CSF MPB leukocytes but not on native leukocytes and not on normal human bone marrow cells. This ~65kDa protein exhibits potent Ca2+-dependent E-selectin ligand activity, as evidenced by (i) binding to fluid phase E-selectin and (ii) avid binding of E-selectin- transfected cells under physiological shear flow conditions. Treatment of normal human bone marrow cells with G-CSF at a pharmacokinetically-relevant concentration in vitro induces the expression of this ~65kDa protein directly on myeloid cells. We hypothesize that this ~65kDa E-selectin ligand contributes to vascular and inflammatory complications following G-CSF administration. The specific aims of this proposal are: (1) To identify the ~65kDa protein serving as an E-selectin ligand on G-CSF MPB leukocytes; and (2) To identify the subset(s) of human leukocyte-series cells that express this glycoprotein following G-CSF administration in vivo and in vitro. The identification and the characterization of expression of this novel E-selectin ligand should provide insights into the biology of E-selectin ligands and greatly increase our understanding of G-CSF MPB leukocyte-endothelial interactions. It is anticipated that the results obtained will lead to new approaches for preventing or alleviating G-CSF-induced vascular and inflammatory complications [unreadable] [unreadable] [unreadable]